Cyclin A2 and Ki-67 proliferation markers could be used to identify tumors with poor prognosis in African American women with breast cancer

Background: Diagnosed invasive breast carcinomas in African American patients are more aggressive compared with those in Caucasian patients and diagnosed at later stages of the disease with higher grade tumors. Despite advances in breast cancer systemic treatment, new prognostic and predictive biomarkers are still needed. Therefore, potential biomarkers were chosen to correlate with different subtypes, recurrence, and survival of invasive breast cancer in a cohort of African American women. Methods: Eight protein biomarkers (ER, PR, HER2, Cyclin A2, Cytokeratin 5, Vimentin, Bcl2, and Ki-67) were evaluated using tissue microarrays (TMAs) and immunohistochemistry (IHC). The IHC results from TMAs were analyzed by both supervised and unsupervised clustering methods. The predictive clusters for the supervised and unsupervised methods were compared for agreement with the empirical classification. Kappa values were used to determine the overall percent correct clusters and agreement between specific clusters. Chi-square statistics was used to examine the association between hierarchical and multinomial logistic clustering methods. Results: Five subtypes of breast tumors with distinct protein expression patterns were identified among the studied 166 breast tumors. Luminal B tumors have been distinguished from luminal A tumors by staining for cell cycle proteins Cyclin A2 and Ki-67, which promote cell proliferation. Forty-nine percent were stained positive for Cyclin A2, 39.2% positive for Ki-67, and 37% positive for both Cyclin A2 and Ki-67. The age of patients did not show any significant effect whether five (p-value= 0.576) or eight (p-value= 0.605) biomarkers were used, which indicating that age did not have any influence on the classification of the subtypes. Ninety percent of the thirty triple negative tumors were positive for Cyclin A2 or Ki-67 or both. Six-year overall survival was better for luminal A tumors (76%) than luminal B tumors (71%). Likewise, six-year relapse-free survival was better for luminal A tumors (76%) than luminal B tumors (29%). Conclusion: Discovery of molecular markers such as Cyclin A2 and Ki-67, and subtypes that are most prevalent in African Americans could lead to a better understanding of the factors contributing to higher morbidity and mortality in this group and to aid in decision-making to offer earlier treatment.


Introduction
Invasive breast carcinoma is the most diagnosed cancer in women in all racial groups in the United States [1]. It is estimated that about 287,850 new cases of invasive breast cancer will be diagnosed in women during 2022 [2]. Breast carcinoma is the leading cause of cancer mortality in African American women [3]. Over the last decades, a paradoxical correlation has been observed among African American women in terms of higher breast cancer mortality rates despite a lower incidence of breast cancer diagnoses compared with Caucasian women [4][5][6]. A greater proportion of breast cancers are diagnosed in African American women of younger age groups compared with Caucasian women (33% of African American women age<50 years vs 25% of Caucasian women) [7,8]. Invasive breast carcinomas diagnosed in African American patients are described as more aggressive compared with those in Caucasian patients and diagnosed at later stages of the disease with higher grade tumors [9,10]. Furthermore, African American women are more likely to have hormone receptive-negative breast cancers than Hispanic women, non-Hispanic White women, and Asian women [11,12]. Moreover, African American cancer patients have shorter survival compared to White women [13].
Several investigators have used tissue microarrays [TMAs] to study the expression of different proteins in breast tissue and other tissues [23][24][25]. TMAs also has been used to identify and/or validate molecular signatures in breast cancer [26][27][28]. TMA immunohistochemistry [IHC] is less expensive, time saving and routinely used in clinical laboratories. Many researchers have used data from TMA of breast cancer samples to correlate with prognosis [21,25,[29][30][31][32][33]. TMA identifies the signature profiles of multiple proteins that are useful for early detection of the primary cancer and recurrence, prediction of survival, identification of tumor subtypes, and drug design. Different scoring systems exist for semi-quantitative evaluation of immunohistochemical staining of breast cancer tissue. Most widely used systems to determine the levels of nuclear, cytoplasmic, and membrane staining in different proteins are the Diallo score [34], Alfred score [35], Histochemical score (H-score) [36] and Quick score [37]. Despite advances in breast cancer systemic treatment, new prognostic and predictive factors are still needed; and the correlation of protein biomarkers with breast cancer subtypes in African Americans could lead to a better understanding of the factors that may be contributing to mortality, prognosis, treatment, and targets for therapy. Therefore, the purpose of this study was to characterize protein markers in African American breast tumors that correspond to different subtypes, recurrence, and survival. recurrence, and survival to characterize protein markers in African American breast cancer tumors. The African American breast cancer patients were diagnosed with an invasive breast ductal carcinomas (IDC) at Howard University Hospital between 2002 and 2009.

Tissue microarrays (TMAs) construction
Triplicate TMAs were constructed in our lab as described by Jacquemier Jocelyne [32], Diallo-Dane Brock [34], and Hewitt SM [38]. Five-micron sections were cut from the archived FFPE blocks, and float mounted on super frost plus micro slides. Slides were stained with hematoxylin-eosin (H&E) and representative areas with IDC were identified and marked on the H&E slides by the pathologist. The individual marked slides were placed on top of each donor block (archived/original FFPE block) and carefully aligned to locate the corresponding tumor sites where the core samples are to be collected. Three separate 1.0 mm tissue cores were obtained from each FFPE donor block and mounted in triplicate recipient TMA blocks using Automated Tissue Arrayer ATA-27 (Beecher Instruments Inc., Pathological Devices, Wisconsin, USA). Spacing between adjacent cores in the recipient TMA blocks was 3 mm. The TMA blocks with core samples were tempered by incubation overnight at 37 °C. Using a microtome, 5-μm sections were cut from the TMA blocks and mounted on Superfrost Plus microscope slides (Thermo Fisher Scientific, MA, USA) for IHC staining.

Immunohistochemical (IHC) staining
The IHC markers were selected based on those known to differentiate breast cancer subtypes with cDNA microarrays, TMA, and protein staining [20,31,[36][37][38]. TMA sections were immune-stained with monoclonal antibodies specific for ER, PR, HER-2, CK5, Vimentin, B-cell lymphoma 2 (Bcl2), Cyclin A2 and Ki-67 (Table 1). The TMA slides were deparaffinized in xylene twice for 5 minutes each, rehydrated with a graded series of alcohol 100%, 95% and 70% ethanol [vol/vol] in deionized water for 5 minutes each. Antigen retrieval for the above selected markers was performed by incubating the slides in Invitrogen 0.01 M citrate buffer (pH 6.0) in a 100°C vegetable steamer (Oster, Sunbeam Products Inc., Boca Raton, Florida, USA) for 20 minutes. Slides were rinsed with phosphate buffer saline (PBS, pH 7.5) at the end of each step in the procedure. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide solution with shaking for 15 minutes at room temperature. Sections were then incubated in unlabeled blocking 1.0% normal goat serum (Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. After that, the slides were incubated at 4°C overnight in a humidified chamber with one of the monoclonal antibodies as described in Table 1. The dilution of each antibody was established based on negative and positive controls and staining with a range of dilutions ( Table 1). The slides were incubated with the appropriate primary antibody and then washed with PBS (pH 7.0) containing 0.1% triton and 0.1% bovine serum albumin. After 30 minutes of incubation at room temperature with a biotinylated goat anti-mouse IgG (0.5%)/ streptavidin complex (VWR, PA, USA), and the positive reaction was visualized using the Avidin Biotin Complex method (ABC kit, Vector Lab). The chromogen substrate was diaminobenzidine (DAB kit, Invitrogen, Waltham, MA, USA). Stained slides were counterstained with hematoxylin (Invitrogen) and finally treated with 70%, 95% and 100% ethanol and xylene. Slides were cover slipped with an automatic unit (Tissue-Tek SCA, Thermo Fisher Scientific) and observed under the light microscope.

Evaluation of immunohistochemical staining
Stained TMA slides were evaluated based on proportion score (%) and relative staining intensity by two pathologists as described by Diallo-Dane Brock [34] and Aaltonen et al. [39]. Slide scores on the bases of proportion of cells stained (extent) and relative staining intensity in the invasive tumor cells present in the tissue core were recorded in Excel file. The Diallo score [34] was used to determine the levels of staining for ER, PR, HER-2, Cyclin A2, Bcl2, CK5, and Vimentin markers. The Diallo scoring system used is as follows: Extent of % stained cells -0 = no cells stained positive; 1 = <25% positive; 2 = 26-50% positive; 3 = 51-75% positive; and 4 = >75% positive. Intensity of stained cells: 0 = negative, 1 = weak staining; 2 = moderate staining; and 3 = strong staining. The intensity and proportion (extent) scores were multiplied to give a final score ranging from 0 to 12.
A sample was judged to be negative with a score of 0 to 3 and positive with score of 4 to 12. The scoring system developed by Cheong et al. [40] and Veronese et al. [41] was used for Ki-67; Ki-67 was interpreted as being increased when >14% of cells show nuclear expression. Membranous IHC staining of +3 or greater for HER-2 was considered positive; +2 scores were re-evaluated by fluorescence in situ hybridization (FISH). Tissue cores that did not adhere to the slide, had no invasive carcinoma, or were otherwise uninterpretable were excluded from scoring and data analysis.

Statistical analyses
Eight markers (ER, PR, HER-2, Cyclin A2, CK5, Vimentin, Bcl2 and Ki-67) were evaluated for identifying breast cancer subtypes in African American women using TMA and IHC. The TMA results were analyzed by both supervised and unsupervised clustering methods. For the unsupervised method, hierarchical clustering of the proteins and their distribution among the 166 FFPE samples were constructed using Cluster 3.0 and Java TreeView software Version 20 [42]. Hierarchical clustering, using complete linkage as cluster method and Euclidean as distance measure [43], was employed to classify samples based on all eight proteins. The NCSS software [44] was used to construct the dendrogram and to show the protein expression profile among the 166 tested breast cancer tumors.
For the supervised method, multinomial logistic regression analysis [45,46] was conducted using an empirically classified training data set. The empirical classification was based on previously published cDNA microarrays, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), TMA, and protein studies [20,32,34,47,48]. The predictive clusters for the supervised and unsupervised methods were compared for agreement with the empirical classification. Kappa values [49] were used to determine the overall percent correct clusters and agreement between specific clusters. For high values of Kappa, the lower limit of the confidence interval was used for verification. Chi-square statistics [50] was used to examine the association between hierarchical and multinomial logistic clustering methods.
The distribution of the 166 breast cancer tumors among the different clusters is shown in Table 3. Most of the tumors belonged to luminal A (30.7%) and triple-negative (29.5%) clusters. The highest frequency of positive immuno-reactivity for Cyclin A2 and Ki-67 was observed in the luminal B and triple-negative subtypes ( Figure 2). The lowest activity of these two markers was in luminal A. Whereas, the expression of Bcl2 was higher in luminal A than luminal B tumors. Therefore, prognostic role of Bcl2 expression in breast cancer is subtype-specific, and Bcl2 expression differs according to the molecular subtype and is a good prognostic marker for only luminal A breast cancer.

Influence of age on the classification of the breast cancer subtypes
The age of patients was analyzed using one-factor ANOVA model and did not show any significant effect whether five (p-value= 0.576) or eight (p-value= 0.605) biomarkers were used, indicating that age did not have any influence on the classification of the subtypes. Thirty-one percent of the patients were less than 50 years old, 30% between 50 to 60 years and 39% older than 60 years (Figure 3).

Dendrogram and sequential hierarchical clustering
The expression profile of the eight biomarkers in the tested breast cancer tissues is shown in Figure 5. Based on the dendrogram, five distinct protein expression profiles are observed: Group 1 consisting of Cyclin A2 and Ki-67; Group 2 consisting of CK5 and Vimentin; Group 3 and Group 4 consisting of HER-2 and Bcl2, respectively; Group 5 consisting of ER and PR. This classification is consistent with the function of the proteins.
The sequential hierarchical clustering shown on the dendrogram in Figure 6 classifies the patient tumors into two Classes 1 and 2 based on ER and PR. Hierarchical clustering using HER-2, Cyclin A2 and Ki-67 classifies Class 1 into clusters 1, 2 and 3; HER-2 by itself classifies Class 2 into clusters 4 and 5. The effect of each of the proteins within the five clusters is also shown in Figure 5. The significant effect of ER frequency is exhibited in clusters 1, 2 and 3. The effect of Cyclin A2 frequency is shown in cluster 1 while that of Ki-67 is shown in clusters 1 and 2. A significant effect of HER-2 frequency is shown for clusters 3 and 4.

Discussion
The existence of clinically distinct breast cancer subtypes has been established based on the protein level by using TMA technology [25,31,36,51]. Previous protein expression profiling studies have reported two to six different numbers of clusters. This variation may be due to the selected marker proteins, the number of breast cancer samples, and the type of used statistical method. Sorlie et al. [15,16] and In the present study, we have identified five subtypes of breast tumors (Luminal A, Luminal B, Luminal B HER-2 + , HER-2, and triple-negative) with distinct protein expression patterns among 166 African American women.
Most studies have applied unsupervised hierarchical clustering method to analyze the IHC scoring data [54][55][56][57]. In our study, we used both the supervised and unsupervised clustering methods and classified the tumor subtypes into ER + and ER − as well as the highly proliferative subgroups. ER and PR clustered together indicating similar function in breast tumor development. The association between ER and PR expression was statistically significant. This significant association between ER and PR in the TMA has also been reported in Danish patients by Henriksen et al. [58] using semi-quantitative IHC analyses. Cyclin A2 and Ki-67 clustered together as proliferation markers. This proliferation cluster is remarkably like that identified by Callogy [47], Korsching [59], and Ameh-Mensah [60] in British, German, and Ghanaian patients; respectively.  (74). Wajid et all reported that expression of ER, PR, and HER-2 resembled that of Western countries with no differences between urban and rural centers of Egypt and most Egyptian cases were classified as Luminal A (44%), which offers the best prognosis of all the subtypes. Elevated Cyclin A2 stimulates oncogenesis by promoting G1/S and G2/M transitions in the cell cycle [75,76]. HER-2 enriched subtype in our study was only 15% of the total 166 patient samples. This subtype is hormone receptor weak/negative, Cyclin A2 positive and shows a high expression of basal type proteins CK5 and Vimentin. In our study, eighty percent (20/25) of the HER-2 + /ER − /PR − tumors were positive for Cyclin A2 and/or Ki-67. According to Morris et al. [11] approximately 30% of breast cancers have an amplification of the HER-2/neu gene or overexpression of its protein product. Overexpression of this receptor in breast cancer is associated with increased disease recurrence and worse prognosis.
According to Pratt et al. [68] many triple-negative tumors were either basal-like (39% to 54%) or Claudin-low (25% to 39%). DNA microarray study by Prat & Peron [69] showed that triple-negative tumors consist of basal (50%), claudin-low (30%), HER-2 + (9%), luminal B (6%) and luminal A (5%) subtypes. The triple-negative group as defined by immunohistochemical staining consists for approximately 80% of basal-like breast cancers as defined by gene expression profiling [77]. Our triple-negative tumors have many of the characteristics of basal tumors: invasive ductal carcinoma, grade 3 tumors, CK5+, Vimentin+, highly proliferative markers (increased Ki-67 + and Cyclin A2 + ). Eighty-four percent (41/49) of the triple-negative (ER − /PR − /HER-2 − ) tumors were also positive for the proliferation markers Cyclin A2 and/or Ki-67. The basal markers CK5 and vimentin were also common in the triple-negative tumors, as expected. Seventy-five percent of the HER-2 + /ER − /PR − tumors were positive for Cyclin A2 and/or Ki-67. The RASSF1A protein has been reported to inhibit the transcription of Cyclin A2 [78]. Methylation of the CpG islands in the RASSF1A promoter is expected to decrease RASSF1A transcription and thereby increase Cyclin A2 transcription and protein levels. We observed significantly increased levels of RASSF1A methylation (%m) with African American triple-negative breast cancer tumors compared to mammoplasty samples (Figure 7). Increased Cyclin A2 protein staining correlated with increased RASSF1A methylation (Figure 7). These data suggest that methylation of RASSF1A is one mechanism by which Cyclin A2 protein levels are elevated in African American breast tumors.
Earlier studies at Howard University Hospital demonstrated that African American triplenegative tumors and HER-2 + /ER − /PR − carcinomas had the poorest breast cancer-specific survival [65]. Furthermore, triple-negative tumors in African American women at Howard University Hospital were significantly associated with a higher incidence of distant metastases [66]. Therefore, it appears that the proliferation proteins, Cyclin A2 and Ki-67, are associated with poor survival among African American breast cancer patients.

Conclusion
We identified five different subtypes with a greater proportion of the patients (60%) belongs to luminal A and triple-negative subtypes. Furthermore, our findings have shown that both Cyclin A2 and Ki-67 proliferation markers can be used for immune-histological identification of breast tumors with poor prognosis. This will aid physicians in making decisions on the treatment of the tumors. Previous studies have demonstrated that proliferation markers are associated with high histological grade and poor prognosis. Our findings are consistent with the previously reported data by other investigators. Therefore, discovery of molecular markers and subtypes that are most prevalent in African Americans could lead to a better understanding of the factors contributing to higher mortality in this group and to better treatment.

Contribution to the Field Statement
It is well established that racial differences can influence breast cancer incidence and mortality. Diagnosed invasive breast carcinomas in African American patients are more aggressive compared with those in Caucasian patients and diagnosed at later stages of the disease with higher grade tumors. African American patients with breast carcinomas are more likely than Caucasian patients to present with tumors that are of a later stage and higher grade, with higher Ki-67 expression and more ER negativity [79].
Earlier studies at Howard University Hospital (HUH) demonstrated that African American triple-negative tumors and HER-2 + /ER − /PR − carcinomas had the poorest breast cancerspecific survival. Furthermore, triple-negative tumors in African American women at HUH were significantly associated with a higher incidence of distant metastases. However, these studies did not differentiate the luminal A tumors from the more aggressive luminal B tumors. Results from our study showed that Cyclin A2 and Ki-67 clearly distinguished luminal A from luminal B. Also, our triple-negative tumors have many of the characteristics of basal tumors: invasive ductal carcinoma, grade 3 tumors, CK5+, Vimentin+, highly proliferative markers (increased Ki-67 + and Cyclin A2 + ). Eighty-four percent of the triple- negative tumors were also positive for the proliferation markers Cyclin A2 and/or Ki-67.
Furthermore, it appears that the proliferation proteins, Cyclin A2 and Ki-67, are associated with poor survival among African American breast cancer patients. Personalized medicine to decrease the adverse effects of chemotherapy is not only requires clinical, but also molecular characterization of tumors, which allows the use of more effective drugs for each patient.
Therefore, both Cyclin A2 and Ki-67 are recommended for immuno-histological identification of tumors with poor prognosis and thus aid physicians in treatment decisions. These data may heighten the need to employ these techs to increase detection rates in this high-risk population.

Limitations
The number of protein biomarkers and patients' samples in the used TMAs for subclassifying tumors into clinically and biologically relevant subgroups were limited. For the study, based on the available resources, we choose to follow the expert panel recommendations and the prognostic significance of Cyclin A2 and Ki-64 in breast cancer mainly in African American women which needs evaluation in larger studies. PAM50 (Prediction Analysis of Microarray 50) molecular testing is not performed on our study population, and we will consider this in our future study to predict the chance of metastasis for some ER-positive, HER2-negative breast cancers. Also, inclusion of CCNA2 immunohistochemistry for the identification of the luminal B subtype.       The sequential hierarchical clustering classifies the patients breast tumors into two Classes 1 and 2 based on ER and PR (using Ki-67 and Cyclin A2 to discriminate between luminal A and luminal B and rather only using HER-2 status). Hierarchical clustering using HER-2, Cyclin A2 and Ki-67 classifies Class 1 into clusters 1, 2 and 3; HER-2 by itself classifies Class 2 into clusters 4 and 5. The effect of each of the proteins within the five clusters is also shown in Figure 5. The significant effect of ER frequency is exhibited in clusters 1, 2 and 3. The effect of Cyclin A2 frequency is shown in cluster 1 while that of Ki-67 is shown in clusters 1 and 2. A significant effect of HER-2 frequency is shown for clusters 3 and 4.  Direct relationship between RASSF1A methylation and Cyclin A2 protein staining in normal and ER-negative/PR-negative/HER2-negative samples. Methylation of the CpG islands in the RASSF1A promoter is expected to decrease RASSF1A transcription and thereby increase Cyclin A2 transcription and protein levels. We observed significantly (p=0.039) increased levels of RASSF1A methylation (%m) in triple negative invasive ductal carcinoma compared to mammoplasty samples in African American (Figure 7, left). Increased Cyclin A2 protein staining correlated with increased RASSF1A methylation (Figure 7, right). These data suggest that methylation of RASSF1A is one mechanism by which Cyclin A2 protein levels are elevated in African American breast cancers. List of the investigated markers and characteristics of the corresponding dilution of the specific anti-human antibody used in immunohistochemistry study. Staining characteristic: expression location in the cell (nuclear, membranous, cytoplasmic). ER: Estrogen; PR: Progesterone; HER-2: Human epidermal growth factor receptor 2; Bcl2: B-cell lymphoma 2; CK5: Cytokine 5. Test of agreement between the hierarchical clustering and multinomial regression for the different subtypes using five markers. The percent agreement between using five and eight biomarkers was 95.8%.